Compositions and methods for the treatment of primary and metastatic neoplastic diseases using arsenic compounds

ABSTRACT

The invention relates to the use of arsenic compounds to treat a variety of neoplastic diseases. The present invention encompasses the administration to a mammal of arsenic in the form of a salt, complex, organic compound or ionic solution to treat tumors of epithelial tissue, connective tissue, central nervous system, lymphoid tissue, hematopoietic cells and tumors associated with oncogenic viruses. This invention also encompasses the treatment of hematopoietic disorders in mammals by the administration of one or more arsenic compounds to said mammal. Further, the arsenic compounds may be used to treat metastatic neoplastic diseases.

1. FIELD OF INVENTION

The present invention relates to methods and compositions for thetreatment of primary and metastatic neoplastic diseases, including, butnot limited to human sarcomas, carcinomas and hematopoietic disorders.In the practice of the treatment of cancer, compositions containingarsenic compounds are used to arrest and reverse neoplastic growth.

More specifically, the present invention relates to novelchemotherapeutic methods—novel uses of arsenic compounds for treatingprimary and metastatic tumors; primary and metastatic tumors of thecentral nervous system; refractory primary and metastatic tumors of thecentral nervous system; breast, lung, bladder and prostate cancer; andrefractory breast, lung, bladder and prostate cancer to mention a few.

2. BACKGROUND OF THE INVENTION

In 1997 more than one million people will develop some type of cancer inthe United States. Approximately 500,000 will be cured or in a state ofremission. These numbers represent an improving cure rate seen over thepast decade which is largely due to earlier detection, better treatmentand advances in chemotherapy. In particular, the advances inchemotherapy include targeted or specific drug therapy in which a drugis developed specifically for the treatment of a certain cancer type.This “disease-oriented” approach is designed to identify compounds whichexert selective effects in vitro on particular tumor types and tofollow-up these leads in vivo utilizing cell lines, (Fiebig et al.,Cancer Treatment Reviews 17:109-117 (1990)). However, the incidence ofcancer is continuing to climb as our population ages and as new cancersdevelop or occur more frequently, such as in patients infected with AIDSvirus. Thus, it is clear that there is a tremendous demand foradditional regimens to treat patients with cancer.

2.1. Pathobiology of Cancer

Cancer is characterized primarily by an increase in the number ofabnormal cells derived from a given normal tissue, invasion of adjacenttissues by these abnormal cells, and lymphatic or blood-borne spread ofmalignant cells to regional lymph nodes and to distant sites(metastasis). Clinical data and molecular biologic studies indicate thatcancer is a multistep process that begins with minor preneoplasticchanges, which may under certain conditions progress to neoplasia.

Pre-malignant abnormal cell growth is exemplified by hyperplasia,metaplasia, or most particularly, dysplasia (for review of such abnormalgrowth conditions, see Robbins and Angell, 1976, Basic Pathology, 2dEd., W. B. Saunders Co., Philadelphia, pp. 68-79). Hyperplasia is a formof controlled cell proliferation involving an increase in cell number ina tissue or organ, without significant alteration in structure orfunction. As but one example, endometrial hyperplasia often precedesendometrial cancer. Metaplasia is a form of controlled cell growth inwhich one type of adult or fully differentiated cell substitutes foranother type of adult cell. Metaplasia can occur in epithelial orconnective tissue cells. Atypical metaplasia involves a somewhatdisorderly metaplastic epithelium. Dysplasia is frequently a forerunnerof cancer, and is found mainly in the epithelia; it is the mostdisorderly form of non-neoplastic cell growth, involving a loss inindividual cell uniformity and in the architectural orientation ofcells. Dysplastic cells often have abnormally large, deeply stainednuclei, and exhibit pleomorphism. Dysplasia characteristically occurswhere there exists chronic irritation or inflammation, and is oftenfound in the cervix, respiratory passages, oral cavity, and gallbladder.

The neoplastic lesion may evolve clonally and develop an increasingcapacity for invasion, growth, metastasis, and heterogeneity, especiallyunder conditions in which the neoplastic cells escape the host's immunesurveillance (Roitt, I., Brostoff, J and Kale, D., 1993, Immunology, 3rded., Mosby, St. Louis, pps. 17.1-17.12).

2.2. AIDS-Related Non-Hodgkin's Lymphoma

Since the discovery of AIDS, the disease has had a close associationwith an interesting spectrum of cancers. Further, the types ofmalignancies and their incidence rates are increasing as the developmentof effective antiretroviral therapies and prophylaxis againstopportunistic infections leads to prolonged survival in theimmunodeficient state for AIDS patients, (Karp and Broder, Cancer Res.51:4747-4756 (1991)). AIDS-related non-Hodgkin's lymphoma was found tooccur in AIDS patients only after 1981. AIDS-related non-Hodgkin'slymphoma is a very aggressive disease with a very high incidence ofcentral nervous system involvement. It is increasing in incidence in theAIDS population. As patients infected with the AIDS virus now livelonger because they are not dying of the usual infections, they aredeveloping lymphoma at an increasing rate. The characteristics ofAIDS-related non-Hodgkin's lymphoma are detailed in an article by Karpand Broder, (1991), supra.

The problems the medical oncologist has in treating patients withAIDS-related lymphomas is the recently described predilection foroccurrence of the lymphoma in the central nervous system (in brain andsurrounding meninges) and the fact that the patient with AIDS has a veryweak bone marrow which cannot tolerate treatment with standardchemotherapy. This makes the treatment of lymphoma in patients with AIDSvery difficult because standard chemotherapeutic agents are usually verymarrow suppressive and do not cross blood brain barrier (to treat thecentral nervous system disease).

2.3. Primary and M Tastatic CNS Tumors

The incidence of primary and metastatic brain tumors is increasing inthe United States. Indeed, the arsenal of chemotherapeutics for thesetypes of cancers is minimal, while the need for such therapeutics ishigh.

Glioblastoma multiform and other primary and metastatic central nervoussystem tumors are devastating malignancies. The treatment of thesetumors include surgery, radiation therapy and treatment with agents suchas the nitrosourea BCNU. Other chemotherapeutic agents utilized includeprocarbazine, vincristine, hydroxyurea and cisplatin. Unfortunately,even when all three modalities (surgery, radiation therapy andchemotherapy) are utilized, the average survival of patients withcentral nervous system malignancies is still about 57 weeks. Clearly,new treatment approaches are needed both for patients with newlydiagnosed primary and metastatic central nervous system tumors, as wellas for patients with such tumors which are refractory to the abovemodalities. Finding such new agents has been complicated by the factthat there is no animal model which appears to predict what agent willbe clinically effective against primary and metastatic central nervoussystem tumors.

2.4. Breast, Lung, Bladder and Prostate Cancers

Breast cancer has been known to occur in about one in every 8-9 women inthe United States. The treatment for early breast cancer is surgery,with or without radiation therapy, or surgery, with or without radiationtherapy, plus chemotherapy and/or hormonal therapy. Despite the bestefforts of physicians there are still more than 80,000 deaths each yearfrom breast cancer and the incidence is still rising. Currentchemotherapy for patients with primary or metastatic breast cancerincludes treatment with cyclophosphamide, methotrexate, doxorubicin,5-fluorouracil, cisplatin, vinblastine, taxol, taxotere, mitomycin C andoccasionally other agents. Unfortunately, even with these agents, almostall women who develop metastatic breast cancer succumb to their disease.One particular place that metastatic breast cancer does metastasize tois the central nervous system. When central nervous system metastases dooccur, the usual treatment is surgery (for a solitary metastasis) orradiation, or surgery plus radiation therapy. At present there is nochemotherapy which is felt to be helpful in this situation.

Lung cancer is responsible for more than 150,000 deaths each year in theUnited States. Most patients with lung cancer present a tumor that hasalready metastasized to a variety of organs, including lung, liver,adrenal gland and other organs. The current treatment for metastaticlung cancer is not yet standardized (Ihde, Daniel C., “Chemotherapy ofLung Cancer”, The New England Journal of Medicine 327:1434-1441, 1992Nov. 12th issue). However, chemotherapy regimens which are utilizedinclude treatment with cisplatin plus etoposide, combinations ofcyclophosphamide plus doxorubicin plus cisplatin, and single agentsalone or in combination, including ifosfamide, teniposide, vindesine,carboplatin, vincristine, taxol, nitrogen mustard, methotrexate,hexamethylmelamine and others. Despite these chemotherapeutic regimensthe average patient with metastatic lung cancer still only survives 7-12months. One particular troublesome place for metastases of lung canceris the central nervous system. The treatment for central nervous systemmetastases includes surgery (to remove a solitary lesion), radiationtherapy, or a combination of both. Unfortunately, there is not standardchemotherapy which is felt to be helpful in this situation.

Each year about 11,000 patients die of bladder cancer in the U.S.Although at presentation the disease is usually localized, most patientsdevelop distant metastatic disease. The most recent advances have beenin the area of chemotherapy for patients with such metastatic disease.One effective regimen is called the MVAC regimen. It consists oftreatment with methotrexate plus vinblastine plus adriamycin(doxorubicin) plus cisplatin. Although the response rate is high to thischemotherapeutic regimen, medical oncologists are noting that one placethe patients fail is with metastases to the central nervous system.Unfortunately, there is no standard chemotherapy which is felt to behelpful in this situation.

It is estimated that more than 100,000 men will be diagnosed withprostate cancer this year and more than 30,000 patients will die fromthe disease. The most common sites of metastases in patients withprostate cancer are the bone and lymph nodes. The bone metastases areparticularly bothersome in that they can create intense pain for thepatient. The current treatment for metastatic prostate cancer includestreatment with flutamide, leuprolide, diethylstilbestrol, and otherhormonal manipulations, as well as chemotherapy (doxorubicin,estramustine phosphate, vinblastine, suramin, cisplatin, and others).Unfortunately, none of these agents are consistently helpful in thedisease. In addition, as patients with prostate cancer live longer withtheir malignancy, they will most likely develop a higher incidence ofmetastases to the central nervous system (including the spinal cord).

In general, as patients are living longer with the common malignanciessuch as breast cancer, lung cancer, bladder cancer, prostate cancer anda variety of other malignancies (because of control of their systemicdisease with surgery, radiation therapy and chemotherapy), oncologistsare noting that they are developing an increasing incidence ofmetastatic tumors in the central nervous system including the brain.This is probably because most of the currently available chemotherapydoes not cross the blood brain barrier. When the patient (who has theirtumor controlled in sites outside of the brain) develops brainmetastases, it is a very difficult situation. Options for that patientare usually limited to surgery for a solitary metastasis and/orradiation therapy. However, after those modalities fail, the patientusually has no other options.

For each of the above indications (primary brain tumors and metastasesto the brain from other common tumors such as breast, lung, bladder andprostate cancers), there is a tremendous need for a more effectivetreatment and/or methods for improving the quality of patient life.

2.5. Esophageal Cancer

In the U.S., carcinoma of the esophagus represents about 6% of allcancers of the gastrointestinal tract but causes a disproportionatenumber of cancer deaths. (Boring, C. C., et al.: Cancer statistics,1993. CA Cancer J. Clin. 43:7, 1993). These cancers usually arise fromthe epithelial layer of the esophagus and are either squamous cellcarcinomas or adenocarcinomas. Overall the 5 year survival is about 5%.

Squamous cell carcinoma generally occurs after the age of 50 and is morecommon in males than in females. The incidence varies widely fromcountry to country and between regions within countries. In the U.S. theincidence is between 2 and 8 persons per 100,000 and is more prevalentin blacks than in whites.

Adenocarcinoma represents 25% of all esophageal CA in the U.S. It isusually located in the distal one third of the esophagus and may invadethe adjacent gastric cardia. It tends to occur in people over 40 yearsof age and is more common in males than in females. It is more common inwhites than in blacks.

2.6. Arsenic and its Medical Uses

Arsenic has been considered to be both a poison and a drug for a longtime in both Western and Chinese medical practices. In the latter partof the nineteenth century, arsenic was used frequently in attempts totreat diseases of the blood in the West. In 1878, it was reported thattreatment of a leukemic patient with Fowler's solution (a solution ofpotassium arsenite) reduced markedly the count of white blood cells(Cutler and Bradford, Am. J. Med. Sci., January 1878, 81-84). Furtherinterests in the use of Fowler's solution as a palliative agent to treatchronic myelogenous leukemia (CML) was described by Forkner and Scott in1931 (J. Am. Med. Assoc., 1931, iii, 97), and later confirmed byStephens and Lawrence in 1936 (Ann. Intern. Med. 9, 1488-1502).Typically, Fowler's solution was orally administered to leukemicpatients as a solution until the level of white blood cells wasdepressed to an acceptable level or until toxicities (such as skinkeratoses and hyperpigmentation) developed, while the patients enjoyedvarying periods of remission. In the 1960's, Fowler's solution was stillused occasionally in attempts to treat CML, however, most patients withCML were treated with other chemotherapeutic agents, such as busulfan,and/or radiation therapy (Monfardini et al., Cancer, 1973, 31:492-501).

Paradoxically, one of the long recognized effects of exposure toarsenic, whether the source is environmental or medicinal, is skincancer (Hutchinson, 1888, Trans. Path. Soc. Lond., 39:352; Neubauer,1947, Br. J. Cancer, 1:192). There were even epidemiological data tosuggest that the use of Fowler's solution over long periods could leadto an increased incidence of cancer at internal sites (Cuzick et al.,Br. J. Cancer, 1982, 45:904-911; Kaspar et al., J. Am. Med. Assoc.,1984, 252:3407-3408). The carcinogenicity of arsenic has since beendemonstrated by the fact that it can induce chromosomal aberration, geneamplification, sister chromatid exchanges and cellular transformation(See e.g., Lee et al., 1988, Science, 241:79-81; and Germolec et al.,Toxicol. Applied Pharmacol., 1996, 141:308-318). Because of the knowncarcinogenic effect of arsenic, its only therapeutic use in human inWestern medicine today is in the treatment of tropical diseases, such asAfrican trypanosomiasis, (melarsoprol, or Arsobal® by Rhône PoulencRorer, Collegeville, Pa.; See Goodman & Gilman's The PharmacologicalBasis of Therapeutics, 9th edition, chapter 66, 1659-1662, 1997).

In traditional chinese medicine, arsenous acid or arsenic trioxide pastehas been used to treat tooth marrow diseases, psoriasis, syphilis andrheumatosis (Chen et al., 1995, in Manual of Clinical Drugs, Shanghai,China, Shanghai Institute of Science and Technology, p. 830). In 1970s,arsenic trioxide had been applied experimentally to treat acutepromyelocytic leukemia (APL) in China (commented by Mervis, 1996,Science, 273:578). The clinical efficacy of arsenic trioxide hasrecently been re-investigated in 14 of 15 patients with refractory APL,where the use of an intravenous dose at 10 mg/day for 4-9 weeks wasreported to result in complete morphologic remission without associatedbone marrow suppression (Shen et al., 1997, Blood, 89:3354-3360). It wasalso reported that arsenic trioxide induced apoptosis (programmed celldeath) in vitro in NB4 cells, an APL cell line, and that apoptosis wasapparently associated with down-regulation of the oncogene bcl-2, andintracellular redistribution of the chimeric PML/RARα protein that areunique to APL cells (Chen et al., 19.96, Blood, 88:1052-1061; Andre etal., 1996, Exp. Cell Res. 229:253-260). Similarly, melarsoprol has beenreported to induce apoptosis in cell lines representative of chronicB-cell leukemia (Konig et al., 1997, Blood 90:562-570). Whetherapoptosis is induced in APL patients is presently unclear, but it isbelieved by some to be one of the possible mechanisms of the therapeuticeffects of certain arsenic compounds.

Although arsenic is well known to be both a poison and a carcinogenicagent, there have been many reports concerning the use of arsenic inmedical treatment. Identification or discussion of the art above mustnot be construed as an admission that such is prior art.

Further, from the above discussion, it should be clear that there are aplethora of different types of cancers, each of which requires a uniquetreatment protocol. Thus, the development of a broad spectrumanti-cancer agent is extremely desirable. At a minimum, additionaleffective anti-cancer agents are needed to be added to the arsenalagainst cancer.

3. SUMMARY OF THE INVENTION

Notwithstanding the conflicting reports in the art concerning benefitsand risks of the administration of arsenic to patients, applicants havediscovered that arsenic compound has broad applicability in thetreatment of various cancers, including solid tumors and blooddisorders. For example, the present invention encompasses the use ofarsenic in the form of a salt, complex, organic compound or ionicsolution to treat tumors of epithelial tissue, connective tissue,central nervous system, lymphoid tissue, hematopoietic cells and tumorsassociated with oncogenic viruses.

Further, the present invention encompasses the use of arsenic compoundsto treat mammals suffering from primary and metastatic neoplasticdisease as well as infectious diseases related thereto.

In addition, this invention also encompasses the use of arseniccompounds to treat primary and metastatic breast, lung, bladder andprostate cancers in humans.

This invention also encompasses the treatment of hematopoietic disordersin mammals by the administration of one or more arsenic compounds tosaid mammal. The hematopoietic disorders to be treated include but arenot limited to polycythemia vera, Hodgkin's Disease, non-Hodgkin'sDisease including Follicular Lymphoma, Diffuse Lymphoma, lymphoblasticlymphoma, small lymphocytic lymphoma, acute lymphocytic leukemia, hairycell leukemia, myeloid metaplasia, myeloid dysplastic syndrome, multiplemyeloma and plasmacytoma.

In accordance with the present invention, arsenic compounds can be usedalone or in combination with other known therapeutic agents (includingchemotherapeutics, radioprotectants and radiotherapeutics) or techniquesto either improve the quality of life of the patient, or to treat theprimary neoplastic disease. For example, the arsenic compounds can beused before, during or after the administration of one or more knownantitumor agents including but not limited to mustard compounds,nitrogen mustard, chlorambucil, melphalan, cyclophosphamide,6-mercaptopurine, 6-thioguanine, cytarabine, 5-fluorouracil,floxuridine, methotrexate, vincristine, vinblastine, taxol, etoposide,temiposide, dactinomycin, daunorubicin, doxorubicin, bleomycin,mitomycin, cisplatin, carboplatin, estramustine phosphate, hydroxyurea,BCNU, procarbazine, VM-26 (vumon), interferons and all-trans retinoicacid (ATRA), (See for example, the Physician Desk References 1997). Inaddition, the arsenic compounds can be used before, during or afterirradiation treatment. For the treatment of HIV-infected individuals,the arsenic compounds can be used alone or in combination with AZT, ddI,ddA, ddC, d4T, 3TC and other known antiviral agents.

The invention described herein encompasses a method of treating primaryand metastatic neoplastic diseases, a method of treating solid tumors, amethod of treating leukemias, a method of treating cancers related tobcl-2 (oncogene), each of which comprises the administration of atherapeutically effective and non-lethal amount of one or more arseniccompounds to a mammal in need of such therapy. The invention, asmentioned above also encompasses the use of combination therapy to treatthe aforementioned diseases.

In a particular embodiment, the arsenic compounds are used within amethod to treat breast, lung, colon, ovarian, renal, non-small celllung, central nervous system, bladder, prostate and head and neck cancerby administering an effective amount of one or more arsenic compoundsalone or in combination with other antineoplastic agents or therapeutictechniques including radiotherapy and surgery.

Without being limited by any theory, the inventors believe that thearsenic compounds of the invention may have one or more mechanisms ofaction in connection with the methods described herein. For example, thearsenic compounds may act as a phosphorous analogue which interfereswith the phosphorylation events that occur in signal transductioninvolved in apoptosis. Arsenic may also act as an inhibitor ofangiogenesis, i.e., the formation of new blood vessels, thereby limitingblood flow to proliferating preneoplastic cell masses, tumors andmetastases. It is well known that if a tumor is not invaded by bloodcapillaries, it would have to depend on the diffusion of nutrients fromits surroundings and cannot enlarge beyond a certain size. Arsenic mayalso function as a differentiating agent which causes dividingpreneoplastic and/or cancer cells that display an undifferentiated orunderdifferentiated phenotype to develop into terminally differentiatedcells, and die after a finite number of cell divisions. Finally, arsenicmay also act to sensitize the cancer cells to radiation and/orchemotherapy. Thus, the arsenic compounds of the invention are describedas being useful against a variety of cancers.

Specific therapeutic regimens, pharmaceutical compositions, and kits arealso provided by the invention. Thus, the invention also encompassespharmaceutical compositions which comprise one or more arsenic compoundsand a pharmaceutically acceptable carrier. The compositions are sterilesolutions suitable for intravenous injection or infusion. In anotherembodiment the invention encompasses a composition suitable for oraldelivery; comprising one or more arsenic compounds and apharmaceutically acceptable excipient or carrier. In another embodiment,the invention also includes compositions suitable for topical or dermaldelivery.

Particular compositions of the invention and their properties aredescribed in the sections and subsections which follow.

4. BRIEF DESCRIPTION OF THE FIGURES

FIG. 1A-1I. Dose response curves showing percentage growth of variouscancer cell lines after continuous exposure to 10⁻⁵ to 10⁻⁹ μg/ml ofarsenic trioxide or 2 days. FIG. 1A. Leukemic cell lines CCRF-CEM,HL-60(TB), K-562, MOLT-4, RPMI-8226, SR. FIG. 1B. Non-Small Cell LungCancer cell lines A549/ATCC, EKVX, HOP-62, HOP-92, NCI-H226, NCI-H23,NCI-8322M, NCI-H460, NCI-H522. FIG. 1C. Colon Cancer cell lines COLO205, HCT-116, HCT-15, HT29, KM12, SW620. FIG. 1D. CNS Cancer cell linesSF-268, SF-295, SF-539, SNB-19, SNB-75, U251. FIG. 1E. Melanoma celllines LOX 1MV1, MALME-3M, M14, SK-MEL-2, SK-MEL-28, SK-MEL-5, UACC-257,UACC-62. FIG. 1F. Ovarian Cancer cell lines IGROV1, OVCAR-3, OVCAR-5,OVCAR-8, SK-OV-3. FIG. 1G. Renal Cancer cell lines A498, CAKI-1, RXE393, SN12C, TX-10, UO-31. FIG. 1H. Prostate Cancer cell lines PC-3,DU-145. FIG. 1I. Breast Cancer cell lines MCF7, NCI/ADR-RES, MDA-MB-435,MDA-N, BT-549, T-47D.

FIG. 2. Mean graphs showing selectivity patterns at each of theprincipal response parameters for all the cell lines tested aftercontinuous exposure to 10⁻⁵ to 10⁻⁹ μg/ml of arsenic trioxide for 2days.

FIG. 3A-3I. Dose response curves showing percentage growth of variouscancer cell lines after continuous exposure to 10⁻⁵ to 10⁻⁹ μg/ml cfarsenic trioxide for 6 days. FIG. 3A. Leukemic cell lines CCRF-CEM,K-562, MOLT-4, RPMI-8226. FIG. 3B. Non-small Cell Lung Cancer cell linesEKVX, HOP-62, HOP-92, NCI-H226, NCI-H23, NCI-H322M, NCI-H460, NCI-H522.FIG. 3C. Colon Cancer cell lines COLO 205, HCT-116, HCT-15, HT29, KM12,SW-620. FIG. 3D. CNS Cancer cell lines SF-268, SF-295, SF-539, SNB-75,U251. FIG. 3E. Melanoma cell lines LOX IMVI, MALMI-3M, SK-MEL-2,SK-MEL-28, SK-MEL-5, UACC-257, UACC-62. FIG. 3F. Ovarian Cancer celllines IGROVI, OVCAR-3, OVCAR-5, OVCAR-8, SK-OV-3. FIG. 3G.

Renal Cancer cell lines 786-0, A498, CAKI-1, RXF 393, S12C, TK-10. FIG.3H. Prostate Cancer cell lines DU-145. FIG. 3I. Breast Cancer cell linesMCF7, NCI/ADR-RIS, MDA-MB-231/ATCC, HS 578T, MDA-MB-435, MDA-N, BR-549,T-47D.

FIG. 4. Mean graphs showing selectivity patterns at each of theprincipal response parameters for all the cell lines tested aftercontinuous exposure to 10⁻⁵ to 10⁻⁹ μg/ml of arsenic trioxide for 6days.

5. DETAILED DESCRIPTION OF THE INVENTION

Methods and compositions for the treatment of primary and metastaticneoplastic diseases are described herein. The invention is based, inpart, on a dosage regimen for administration of compositions comprisingarsenic. The invention is also based in part, on the potency of thearsenic compounds of the invention against certain cancers.

This invention includes a method of treating primary solid tumors in amammal which comprises administering to a mammal in need of such therapya therapeutically effective and non-lethal amount of one or more arseniccompound.

The invention also includes a method of treating metastatic tumors in amammal which comprises administering to a mammal a therapeuticallyeffective and non-lethal dose of one or more arsenic compound.

The invention includes a method for treating disorders of blood inmammal which comprises administering one or more arsenic compound in atherapeutically effective and non-lethal amount.

The arsenic compound of the invention may be utilized in in a variety ofknown forms; for example, arsenic can be administered as a salt, anorganic or inorganic complex, an organic chelate, an organic compound oran organic or inorganic solution. It is preferred that the form bechosen to reduce toxicity and improve efficacy. Further, the form chosenmay also depend on the type and location of the tumor in question. Theinorganic salt forms of arsenic are preferred. For example, inorganicsalts such as arsenic triiodide, arsenic (III) bromide, arsenic (III)chloride, arsenic pentoxide, arsenic trioxide, Fowler's solution(potassium arsenite), sodium arsenite, and calcium arsenite may be used.Arsenic trioxide is most preferred. Both arsenous acids and arsenites aswell as arsenic acids and arsenates may be used within the presentmethods. Aqueous solutions containing arsenite ions are preferred.Further, arsenic sulfides may be used such as arsenous sulfide, arsenicsulfide, arsenic pentasulfide, tetraarsenic trisulfide and tetraarsenicpentasulfide. Without being limited by any theory, certain of thesearsenic compounds may be prodrugs to an active species.

Generally, the skilled artisan will recognize that the form of arsenicto be used should be therapeutically effective without unreasonabletoxicity. The toxicity is dependent upon the dose, the dosage form, themode of administration and frequency of dosing. Generally, the skilledartisan can chose from the following known forms of arsenic: arsenichalides, arsenic oxides, arsenic acids, arsenic sulfides, and the like.

Arsenic can also be readily combined with carbon to form a wide varietyof organic compounds. These include but are not limited to primary andsecondary arsines, tertiary arsines, halo arsines, dihalo arsines,cyclic and polymeric substances containing arsenic; specific examples oforganic arsenic compounds include but are not limited to3-Nitro-4-hydroxyphenylarsonic acid, arsanilic acid, sodium hydrogen4-aminophenylarsenate, melarsoprol, melarsonyl potassium, carbarsone,arsenamide arsphenamine and sodium arsanilate.

As used herein, “arsenic compound” refers to a pharmaceuticallyacceptable form of arsenic including salts, solutions, complexes,chelates and organic and inorganic compounds incorporating arsenic. Itshould be recognized that the invention includes arsenic prodrugs orcompounds that are converted in vivo to biologically active forms ofarsenic. Such prodrugs may be used to reduce or avoid the well knownpotential for arsenic toxicity. The arsenic compounds of the presentinvention can be synthesized or commercially purchased. For example, thecompounds can be prepared from well-known chemical techniques. (See forexample, Kirk-Othmer, Encyclopedia of Chemical Technology 4 ed. volume 3pps. 633-655 John Wiley & Sons).

In one embodiment, the arsenic compound of the invention is arsenictrioxide which is dissolved in an aqueous solution of sodium hydroxide,with the pH adjusted to a physiologically acceptable range, e.g. aboutpH 6-8.

Any suitable mode of administration may be used in accordance with thepresent invention including but not limited to parenteral administrationsuch as intravenous, subcutaneous, intramuscular and intrathecaladministration; oral, intranasal, rectal or vaginal administration mayalso be used; directly into the tumor; transdermal patches; implantdevices (particularly for slow release); finally, topical administrationmay be used. The mode of administration will vary according to the typeof arsenic compound being used and the disease to be treated.

The pharmaceutical compositions to be used may be in the form of sterilephysiologically acceptable (aqueous or organic) solutions, colloidalsuspensions creams, ointments, pastes, capsules, caplets, tablets andcachets. The pharmaceutical compositions comprising arsenic compounds ofthe invention can be contained in sealed sterile glass containers and/orampoules. Further, the active ingredient may be micro-encapsulated,encapsulated in a liposome, noisome or lipofoam alone or in conjunctionwith targeting antibodies. It should be recognized that delayed slow orsustained release forms of administration are also included.

The arsenic compounds of the present invention may be used against avariety of primary and metastatic neoplastic diseases including but notlimited to primary and metastatic tumors of the central nervous system,breast, colon, ovaries, kidneys, lung, bladder, prostate and head andneck.

More specifically, the arsenic compounds of the present invention can beused to treat tumors of epithelial origin including but not limited to:

-   -   squamous cell carcinoma    -   basal cell carcinoma    -   melanoma        tumors of epithelial lining of glands or ducts:    -   adenocarcinoma    -   papillary carcinoma    -   papillary adenocarcinoma        tumors of the liver and biliary tract:    -   Hepatocellular carcinoma        tumors of the gastrointestinal tract:    -   squamous cell carcinoma of the esophagus    -   adenocarcinoma of the esophagus    -   colorectal carcinoma (colon cancer)    -   gastric carcinoma (stomach cancer)        tumors of respiratory tract:    -   bronchogenic carcinoma    -   small cell carcinoma    -   large cell carcinoma        tumors of the urogenital tract:    -   transitional cell carcinomas of bladder    -   squamous cell carcinoma of bladder    -   carcinoma of prostate    -   carcinoma of cervix        tumors of breast        tumors of blood cells and related cells (leukemias):    -   acute and chronic lymphocytic leukemia    -   polycythemia vera        Cancers of Lymphoid tissue    -   Malignant Lymphomas-Hodgkins Lymphoma    -   Non-Hodgkin's Lymphoma-Follicular lymphoma        -   Diffuse lymphoma        -   Small lymphocytic lymphoma    -   Large cell lymphoma    -   Lymphoblastic lymphoma    -   Multiple myeloma        Tumors of Connective Tissue        Cancers of Bone    -   Osteosarcoma        Tumors of the Nervous System    -   Neuroblastoma    -   Retinoblastoma    -   Glioblastoma    -   Oligodendroglioma        Tumors associated with oncogenic viruses    -   Human Papillomavirus-squamous cell carcinoma of cervix    -   Ebstein-Barr Virus-Burkitts Lymphoma        -   B cell lymphoma's in        -   immuno-comprised individuals        -   Nasopharyngeal carcinoma    -   Hepatitis B Virus-Hepatocellular carcinoma

Herpes Virus 8 or Kaposi Sarcoma Herpes Virus (KSHV)—Kaposi's Sarcoma,and the like. Other neoplastic diseases known to the skilled artisan arealso encompassed by the present invention including cancer of the oralcavity, larynx, kidney, testis and ovary. The skilled artisan willrecognize that other cancers may be treated in accordance with thepresent invention.

The term “a method for treating primary and metastatic tumors of thecentral nervous system” as used herein means that the disease and thesymptoms associated with the disease are alleviated, reduced, cured, orotherwise placed in a state of remission.

As used herein, the terms “a method for treating primary or metastaticbreast, lung, bladder or prostate cancer” and “a method for treatingmetastases from breast, lung, bladder or prostate cancer” means that thedisease and the symptoms associated with the disease are alleviated,reduced, cured, or placed in a state of remission. In addition, the term“a method for treating metastases from breast, lung, bladder or prostatecancer” means that the metastatic tumors and the symptoms associatedwith the disease are alleviated, reduced, cured, placed in a state ofremission.

The term “refractory” when used herein means that malignancies aregenerally resistant to treatment or cure. The term “refractory” whenused in the above terms, means that the malignancies which are generallyresistant to treatment or cure are alleviated, reduced, cured, or placedin a state of remission.

As used herein the terms “a therapeutic agent”, “therapeutic regimen”,“radioprotectant”, “chemotherapeutic” mean conventional drugs and drugtherapies, including vaccines, for treating cancer, viral infections,and other malignancies, which are known to those skilled in the art.“Radiotherapeutic” agents are well known in the art.

As used herein, “a method of treating cancer” or “a method of treatingsolid tumors” or “a method of treating neoplastic diseases” means thatthe disease and the symptoms associated with the disease are alleviated,reduced, cured, or placed in a state of remission. Further, tumor growthis inhibited and/or tumor size is reduced.

As used herein, “preneoplastic” cell refers to a cell which is intransition from a normal to a neoplastic form; and morphologicalevidence, increasingly supported by molecular biologic studies,indicates that preneoplasia progresses through multiple steps.Non-neoplastic cell growth commonly consists of hyperplasia, metaplasia,or most particularly, dysplasia (for review of such abnormal growthconditions (See. Robbins and Angell, 1976, Basic Pathology, 2d Ed., W.B. Saunders Co., Philadelphia, pp. 68-79). Hyperplasia is a form ofcontrolled cell proliferation involving an increase in cell number in atissue or organ, without significant alteration in structure orfunction. As but one example, endometrial hyperplasia often precedesendometrial cancer. Metaplasia is a form of controlled cell growth inwhich one type of adult or fully differentiated cell substitutes foranother type of adult cell. Metaplasia can occur in epithelial orconnective tissue cells. Atypical metaplasia involves a somewhatdisorderly metaplastic epithelium. Dysplasia is frequently a forerunnerof cancer, and is found mainly in the epithelia; it is the mostdisorderly form of non-neoplastic cell growth, involving a loss inindividual cell uniformity and in the architectural orientation ofcells. Dysplastic cells often have abnormally large, deeply stainednuclei, and exhibit pleomorphism. Dysplasia characteristically occurswhere there exists chronic irritation or inflammation, and is oftenfound in the cervix, respiratory passages, oral cavity, and gallbladder. Although preneoplastic lesions may progress to neoplasia, theymay also remain stable for long periods and may even regress,particularly if the inciting agent is removed or if the lesion succumbsto an immunological attack by its host.

The therapeutic regimens and pharmaceutical compositions of theinvention may be used with additional immune response enhancers orbiological response modifiers including, but not limited to, thecytokines IFN-α, IFN-γ, IL-2, IL-4, IL-6, TNF, or otherimmunostimulatants/immunomodulators. In accordance with this aspect ofthe invention, the arsenic compounds are administered in combinationtherapy with one or more of these agents.

5.1. Formulation

The arsenic compounds of the invention may be formulated intopharmaceutical preparations for administration to mammals for treatmentof cancer. Compositions comprising a compound of the inventionformulated in a compatible pharmaceutical carrier may be prepared,packaged, labelled for treatment of and used for the treatment of theindicated tumor, such as human sarcomas and carcinomas, e.g.,fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteogenicsarcoma, chordoma, angiosarcoma, endotheliosarcoma, lymphangiosarcoma,lymphangioendotheliosarcoma, synovioma, mesothelioma, Ewing's tumor,leiomyosarcoma, rhabdomyosarcoma, colon carcinoma, pancreatic cancer,breast cancer, ovarian cancer, prostate cancer, squamous cell carcinoma,basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, sebaceousgland carcinoma, papillary carcinoma, papillary adenocarcinomas,cystadenocarcinoma, medullary carcinoma, bronchogenic carcinoma, renalcell carcinoma, hepatoma, bile duct carcinoma, choriocarcinoma,seminoma, embryonal carcinoma, Wilms' tumor, cervical cancer, testiculartumor, lung carcinoma, small cell lung carcinoma, bladder carcinoma,epithelial carcinoma, glioma, astrocytoma, medulloblastoma,craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, acousticneuroma, oligodendroglioma, meningioma, melanoma, neuroblastoma,retinoblastoma; leukemias, e.g., acute lymphocytic leukemia(myeloblastic, promyelocytic, myelomonocytic, monocytic anderythroleukemia); chronic leukemia and chronic lymphocytic leukemia; andpolycythemia vera, lymphoma (Hodgkin's disease and non-Hodgkin'sdisease), multiple myeloma, Waldenström's macroglobulinemia, and heavychain disease. Alternatively, it can be labeled for treatment of theappropriate infectious disease. Alternatively, pharmaceuticalcompositions may be formulated for treatment of appropriate infectiousdiseases.

If the complex is water-soluble, then it may be formulated in anappropriate buffer, for example, phosphate buffered saline or otherphysiologically compatible solutions. Alternatively, if the resultingcomplex has poor solubility in aqueous solvents, then it may beformulated with a non-ionic surfactant such as Tween, polyethyleneglycol or glycerine. Thus, the compounds and their physiologicallyacceptable solvates may be formulated for administration by inhalationor insufflation (either through the mouth or the nose) or oral, buccal,parenteral, topical, dermal, vaginal, drug delivery device, e.g., porousor viscous material such as lipofoam, rectal administration or, in thecase of tumors, directly injected into a solid tumor.

For oral administration, the pharmaceutical preparation may be in liquidform, for example, solutions, syrups or suspensions, or may be presentedas a drug product for reconstitution with water or other suitablevehicle before use. Such liquid preparations may be prepared byconventional means with pharmaceutically acceptable additives such assuspending agents (e.g., sorbitol syrup, cellulose derivatives orhydrogenated edible fats); emulsifying agents (e.g., lecithin oracacia); non-aqueous vehicles (e.g., almond oil, oily esters, orfractionated vegetable oils); and preservatives (e.g., methyl orpropyl-p-hydroxybenzoates or sorbic acid). The pharmaceuticalcompositions may take the form of, for example, tablets or capsulesprepared by conventional means with pharmaceutically acceptableexcipients such as binding agents (e.g., pregelatinized maize starch,polyvinyl pyrrolidone or hydroxypropyl methylcellulose); fillers (e.g.,lactose, microcrystalline cellulose or calcium hydrogen phosphate);lubricants (e.g., magnesium stearate, talc or silica); disintegrants(e.g., potato starch or sodium starch glycolate); or wetting agents(e.g., sodium lauryl sulphate). The tablets may be coated by methodswell-known in the art.

Preparations for oral administration may be suitably formulated to givecontrolled release of the active compound.

For buccal administration, the compositions may take the form of tabletsor lozenges formulated in conventional manner.

For administration by inhalation, the compounds for use according to thepresent invention are conveniently delivered in the form of an aerosolspray presentation from pressurized packs or a nebulizer, with the useof a suitable propellant, e.g., dichlorodifluoromethane,trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide orother suitable gas. In the case of a pressurized aerosol the dosage unitmay be determined by providing a valve to deliver a metered amount.Capsules and cartridges of, e.g., gelatin for use in an inhaler orinsufflator may be formulated containing a powder mix of the compoundand a suitable powder base such as lactose or starch.

The compounds may be formulated for parenteral administration byinjection, e.g., by bolus injection or continuous infusion. Suchformulations are sterile.

Formulations for injection may be presented in unit dosage form, e.g.,in ampules or in multi-dose containers, with an added preservative. Thecompositions may take such forms as suspensions, solutions or emulsionsin oily or aqueous vehicles, and may contain formulatory agents such assuspending, stabilizing and/or dispersing agents. Alternatively, theactive ingredient may be in powder form for constitution with a suitablevehicle, e.g., sterile pyrogen-free water, before use.

The compounds may also be formulated in rectal compositions such assuppositories or retention enemas, e.g., containing conventionalsuppository bases such as cocoa butter or other glycerides.

In addition to the formulations described previously, the compounds mayalso be formulated as a depot preparation. Such long acting formulationsmay be administered by implantation (for example, subcutaneously orintramuscularly) or by intramuscular injection. Thus, for example, thecompounds may be formulated with suitable polymeric or hydrophobicmaterials (for example, as an emulsion in an acceptable oil) or ionexchange resins, or as sparingly soluble derivatives, for example, as asparingly soluble salt. Liposomes and emulsions are well known examplesof delivery vehicles or carriers for hydrophilic drugs.

The compositions may, if desired, be presented in a pack or dispenserdevice which may contain one or more unit dosage forms containing theactive ingredient. The pack may for example comprise metal or plasticfoil, such as a blister pack. The pack or dispenser device may beaccompanied by instructions for administration.

The invention also provides kits for carrying out the therapeuticregimens of the invention. Such kits comprise in one or more containerstherapeutically effective amounts of the arsenic compounds inpharmaceutically acceptable form. The arsenic compound in a vial of akit of the invention may be in the form of a pharmaceutically acceptablesolution, e.g., in combination with sterile saline, dextrose solution,or buffered solution, or other pharmaceutically acceptable sterilefluid. Alternatively, the complex may be lyophilized or desiccated; inthis instance, the kit optionally further comprises in a container apharmaceutically acceptable solution (e.g., saline, dextrose solution,etc.), preferably sterile, to reconstitute the complex to form asolution for injection purposes.

In another embodiment, a kit of the invention further comprises a needleor syringe, preferably packaged in sterile form, for injecting thecomplex, and/or a packaged alcohol pad. Instructions are optionallyincluded for administration of arsenic compounds by a clinician or bythe patient.

The magnitude of a therapeutic dose of an arsenic compound in the acuteor chronic management of cancer will vary with the severity of thecondition to be treated and the route of administration. The dose, andperhaps dose frequency, will also vary according to the age, bodyweight, condition and response of the individual patient. In general,the total daily dose ranges for the conditions described herein aregenerally from about 10 μg to about 200 mg administered in divided dosesadministered parenterally or orally or topically. A preferred totaldaily dose is from about 0.5 mg to about 70 mg of the active ingredient.

Desirable blood levels may be maintained by a continuous infusion of anarsenic compound-as ascertained by plasma levels. It should be notedthat the attending physician would know how to and when to terminate,interrupt or adjust therapy to lower dosage due to toxicity, or bonemarrow, liver or kidney dysfunctions. Conversely, the attendingphysician would also know how to and when to adjust treatment to higherlevels if the clinical response is not adequate (precluding toxic sideeffects).

Again, any suitable route of administration may be employed forproviding the patient with an effective dosage of an arsenic compound.For example, oral, rectal, vaginal, transdermal, parenteral(subcutaneous, intramuscular, intrathecal and the like) may be employed.Dosage forms include tablets, troches, cachet, dispersions, suspensions,solutions, capsules, patches, and the like. (See, Remington'sPharmaceutical Sciences.)

The pharmaceutical compositions of the present invention comprise anarsenic compound as the active ingredient, or a pharmaceuticallyacceptable salt thereof, and may also contain a pharmaceuticallyacceptable carrier, and optionally, other therapeutic ingredients, forexample antivirals. The term “pharmaceutically acceptable salts” refersto salts prepared from pharmaceutically acceptable non-toxic acids andbases, including inorganic and organic acids and bases.

The pharmaceutical compositions include compositions suitable for oral,rectal, mucosal routes, transdermal, parenteral (including subcutaneous,intramuscular, intrathecal and intravenous), although the most suitableroute in any given case will depend on the nature and severity of thecondition being treated.

In the case where an intravenous injection or infusion composition isemployed, a suitable dosage range for use is, e.g., from about 0.5 mg toabout 150 mg total daily dose.

In addition, the arsenic carrier could be delivered via charged anduncharged matrices used as drug delivery devices such as celluloseacetate membranes, also through targeted delivery systems such asfusogenic liposomes attached to antibodies or specific antigens.

In practical use, an arsenic compound can be combined as the activeingredient in intimate admixture with a pharmaceutical carrier accordingto conventional pharmaceutical compounding techniques. The carrier maytake a wide variety of forms depending on the form of preparationdesired for administration, e.g., oral or parenteral (including tablets,capsules, powders, intravenous injections or infusions). In preparingthe compositions for oral dosage form any of the usual pharmaceuticalmedia may be employed, e.g., water, glycols, oils, alcohols, flavoringagents, preservatives, coloring agents, and the like; in the case oforal liquid preparations, e.g., suspensions, solutions, elixirs,liposomes and aerosols; starches, sugars, micro-crystalline cellulose,diluents, granulating agents, lubricants, binders, disintegratingagents, and the like in the case of oral solid preparations e.g.,powders, capsules, and tablets. In preparing the compositions forparenteral dosage form, such as intravenous injection or infusion,similar pharmaceutical media may be employed, e.g., water, glycols,oils, buffers, sugar, preservatives and the like know to those skilledin the art. Examples of such parenteral compositions include, but arenot limited to Dextrose 5% w/v, normal saline or other solutions. Thetotal dose of the arsenic compound may be administered in a vial ofintravenous fluid, e.g., ranging from about 2 ml to about 2000 ml. Thevolume of dilution fluid will vary according the total doseadministered.

5.2. Target Cancers

Cancers that can be treated by the methods of the present inventioninclude, but not limited to human sarcomas and carcinomas, e.g.,fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteogenicsarcoma, chordoma, angiosarcoma, endotheliosarcoma, lymphangiosarcoma,lymphangioendotheliosarcoma, synovioma, mesothelioma, Ewing's tumor,leiomyosarcoma, rhabdomyosarcoma, colon carcinoma, pancreatic cancer,breast cancer, ovarian cancer, prostate cancer, squamous cell carcinoma,basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, sebaceousgland carcinoma, papillary carcinoma, papillary adenocarcinomas,cystadenocarcinoma, medullary carcinoma, bronchogenic carcinoma, renalcell carcinoma, hepatoma, bile duct carcinoma, choriocarcinoma,seminoma, embryonal carcinoma, Wilms' tumor, cervical cancer, testiculartumor, lung carcinoma, small cell lung carcinoma, bladder carcinoma,epithelial carcinoma, glioma, astrocytoma, medulloblastoma,craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, acousticneuroma, oligodendroglioma, meningioma, melanoma, neuroblastoma,retinoblastoma; leukemias, e.g., acute lymphocytic leukemia(myeloblastic, myelomonocytic, monocytic and erythroleukemia); andchronic lymphocytic leukemia; and polycythemia vera, lymphoma (Hodgkin'sdisease and non-Hodgkin's disease), multiple myeloma, Waldenströmsmacroglobulinemia, and heavy chain disease. Specific examples of suchcancers are described in the sections below.

In a specific embodiment the cancer is metastatic. In another specificembodiment, the patient having a cancer is immunosuppressed by reason ofhaving undergone anti-cancer therapy (e.g., chemotherapy radiation)prior to administration of the arsenic compounds of the invention.

In a specific embodiment, the present invention provides compositionsand methods for enhancing tumor specific immunity in individualssuffering from colorectal cancer metastasized to the liver, in order toinhibit the progression of the neoplastic disease. Preferred methods oftreating these neoplastic diseases comprise administering a compositionof arsenic which elicits an immune response against tumor cells.

In another specific embodiment, the present invention providescompositions and methods for enhancing specific immunity in individualssuffering from hepatocellular carcinoma in order; to inhibit theprogression of the neoplastic disease and ultimately; irradiate allpreneoplastic and neoplastic cells.

In a specific embodiment, the present invention provides compositionsand methods for enhancing specific immunity to preneoplastic andneoplastic mammary cells in women. The present invention also providescompositions and methods for inhibiting cancer cell proliferation andmetastasis. These compositions can be applied alone or in combinationwith each other or with biological response modifiers.

6. WORKING EXAMPLES

The following subsections describe the testing of a pharmaceuticalcomposition comprising arsenic trioxide in vitro using a panel of cancercell lines employed by the National Cancer Institute (NCI). The resultsdemonstrate that arsenic trioxide is effective in inhibiting the growthof a broad range of leukemic cells and cancer cells in vitro.

6.1. METHODS AND MATERIALS

Arsenic trioxide (1 mg/ml, 10 mg/ampoule, manufactured by TaylorPharmaceuticals, Decatur, Ill.) was tested at five concentrations eachat 10-fold dilutions, i.e., 10⁻⁵, 10⁻⁶, 10⁻⁷, 10⁻⁸, and 10⁻⁹ g/ml.

The in vitro tests were performed by incubating the test cells in thepresence of the indicated concentration of arsenic trioxide understandard culture conditions for a designated period of tame, which isfollowed by a sulforhodamine B (SRB) protein assay to estimate cellviability or growth. The cell lines are organized into subpanelsaccording to the origin of the cell lines, e.g., leukemia, breastcancer, etc. A description of the cell lines and method of testing isdescribed in Monk et al. (1997, Anticancer Drug Des. 12:533-41) andWeinstein et al., (1997, Science 275:343-9), which are incorporatedherein in their entirety.

Described below are the data analysis procedures and displays.

The measurement of an effect is expressed in Percentage Growth (PG). Themeasured effect of the compound on a cell line is calculated accordingto one or the other of the following two expressions:If (Mean OD_(test)−Mean OD_(zero))≧0. then PG=100×(Mean OD_(test)−MeanOD_(zero))/(Mean OD_(ctrl)−Mean OD_(zero))If (Mean OD_(test)−Mean OD_(zero))<0. then PG=100×(Mean OD_(test)−MeanOD_(zero))/Mean OD_(zero)where:

-   -   Mean OD_(zero) the average of optical density measurements of        SRB-derived color just before exposure of cells to the test        compounds;    -   Mean OD_(test)=the average of optical density measurements of        SRB-derived color after exposure of cells to the test compound        for a designated period of time; and    -   Mean OD_(ctrl)=the average of optical density measurements of        SRB-derived color after with no exposure of cells to the test        compound for a designated period of time.

Table 1 and 2 present the experimental data collected against each cellline. The first two columns describe the subpanel (e.g., leukemia) andcell line (e.g., CCRF-CEM) involved. The next two columns list the MeanOD_(zero) and Mean OD_(ctrl); the next five columns list the MeanOD_(test) for each of five different concentrations. Each concentrationis expressed as the log₁₀ (molar or μg/ml). The next five columns listthe calculated PGs for each concentration. The response parameters GI50,TGI and LC50 are interpolated values representing the concentrations atwhich the PG is +50, 0, and −50, respectively. Sometimes these responseparameters cannot be obtained by interpolation. If, for instance, all ofthe PGs in a given row exceed +50, then none of the three parameters canbe obtained by interpolation. In such a case, the value given for eachresponse parameter is the highest concentration tested and is precededby a “>” sign. This practice is extended similarly to the other possiblesituations where a response parameter cannot be obtained byinterpolation.

A dose-response curve (see FIGS. 1A-1I and 3A-3I) for the set of data iscreated by plotting the PGs against the log₁₀ of the correspondingconcentration for every cell line. The cell line curves are grouped bysubpanel. Horizontal lines are provided at the PG value of +50, 0, and−50. The concentrations corresponding to points where the curves crossthese lines are the GI50, TGI, and LC50, respectively.

A mean graph (FIGS. 2 and 4) facilitates visual scanning of data forpotential patterns of selectively for particular cell lines or forparticular subpanels with respect to a selected response parameter.Differences in apparent selectivity patterns may occur for the samecompound against the same cell lines when different parameters arecompared. The mean graphs page of the data package shows mean graphs ateach of the principal response parameters: GI50, TGI, and LC50. Barsextending to the right represent sensitivity of the cell line to thetest agent in excess of the average sensitivity of all tested celllines. Since the bar scale is logarithmic, a bar 2 units to the rightimplies the compound achieved the response parameter (e.g., GI50) forthe cell line at a concentration one-hundredth the mean concentrationrequired over all cell lines, and thus the cell line is unusuallysensitive to that compound. Bars extending to the left correspondinglyimply sensitivity less than the mean. If for a particular drug and cellline, it was not possible to determine the desired response parameter byinterpolation, the bar length shown is either the highest concentrationtested (and the listed log₁₀ of the response parameter will be precededby a “>”) or the lowest concentration tested (and the listed log₁₀ willbe preceded by a “1<”).

The values at either limit (> or <) are also calculated in the mean usedfor the mean graph. Therefore, the mean used in the mean graph may notbe the actual mean of the GI50 for instance. For this reason, this valueis referred to as the MG_MID (for mean graph midpoint).

6.2. RESULTS

The results of two sets of tests is presented below. In the first set,the cells from 56 different cancer cell lines were exposed to fiveconcentrations of arsenic trioxide continuously for two days prior toperforming the SRB assay. In the second set, the cells from 50 differentcell lines (a subset of the first 56 cell lines, plus the renal cancercell line 786-0) were exposed continuously for six days prior to the SRBassay. TABLE 1 Log10 Concentration Mean Optical Densities Percent GrowthTime Zero Ctrl −8.9 −7.9 −6.9 −5.9 −4.9 −8.9 −7.9 −6.9 −5.9 −4.9 GI50TGI LC50 Leukemia CCRF-CEM 0.300 1.155 1.203 1.195 1.134 0.704 0.285 106105 98 47 −5  1.11E−06  1.00E−05 >1.26E−05 HL-60(TB) 0.233 0.530 0.5330.507 0.535 0.499 0.213 101 92 102 90 −9  3.19E−06  1.03E−05 >1.26E−05K-562 0.209 1.416 1.387 1.431 1.418 1.124 0.199 98 101 100 76 −5 2.63E−06  1.10E−05 >1.26E−05 MOLT-4 0.134 0.438 0.465 0.454 0.454 0.3680.146 109 105 105 77 4  2.96E−06 >1.26E−05 >1.26E−05 RPMI-8226 0.2570.893 0.868 0.848 0.813 0.670 0.204 96 93 87 65 −21  1.88E−06 7.23E−06 >1.26E−05 SR 0.158 0.457 0.454 0.425 0.457 0.338 0.111 99 89100 60 −30  1.63E−06  5.85E−06 >1.26E−05 Non-Small Cell Lung CancerA549/ATCC 0.015 0.477 0.479 0.486 0.476 0.516 0.336 100 102 100 10869 >1.26E−05 >1.26E−05 >1.26E−05 EKVX 0.342 0.736 0.809 0.849 0.8410.853 0.385 119 129 127 130 11  5.91E−06 >1.26E−05 >1.26E−05 HOP-620.335 1.109 1.088 1.099 1.113 1.086 0.605 97 99 100 97 35 7.18E−06 >1.26E−05 >1.26E−05 HOP-92 0.505 1.694 1.554 1.603 1.477 1.3810.873 88 92 82 74 31  4.50E−06 >1.26E−05 >1.26E−05 NCI-H226 0.560 0.9320.967 0.918 0.967 0.967 0.904 109 96 109 10992 >1.26E−05 >1.26E−05 >1.26E−05 NCI-H23 0.648 1.622 1.769 1.822 1.8801.635 1.215 115 121 127 101 58 >1.26E−05 >1.26E−05 >1.26E−05 NCI-8322M0.382 0.997 1.103 1.036 0.976 0.992 0.755 117 106 97 9961 >1.26E−05 >1.26E−05 >1.26E−05 NCI-H460 0.296 1.235 1.132 1.186 1.2341.157 0.757 89 95 100 92 49  1.20E−05 >1.26E−05 >1.26E−05 NCI-H522 0.4781.138 1.332 1.135 1.189 0.892 0.378 129 100 108 63 −21  1.79E−06 7.08E−06 >1.26E−05 Colon Cancer COLO 205 0.328 1.394 1.425 1.414 1.5761.434 0.935 103 102 117 104 57 >1.26E−05 >1.26E−05 >1.26E−05 HCT-1160.301 1.574 1.508 1.480 1.488 1.391 0.685 95 93 93 86 30 5.53E−06 >1.26E−05 >1.26E−05 HCT-15 0.219 1.623 1.800 1.627 1.673 1.5220.504 113 100 104 93 20  4.90E−06 >1.26E−05 >1.26E−05 HT29 0.095 0.5780.637 0.599 0.580 0.479 0.169 112 104 100 80 15 3.63E−06 >1.26E−05 >1.26E−05 KM12 0.189 0.741 0.744 0.728 0.777 0.7370.567 101 98 106 99 68 >1.26E−05 >1.26E−05 >1.26E−05 SW-620 0.153 0.8860.898 0.868 0.857 0.779 0.267 102 98 96 85 16 4.05E−06 >1.26E−05 >1.26E−05 CNS Cancer SF-268 0.203 0.767 0.821 0.7360.815 0.767 0.334 110 94 108 100 23  5.63E−06 >1.26E−05 >1.26E−05 SF-2950.249 1.007 0.951 0.978 0.938 0.938 0.639 93 96 91 9151 >1.26E−05 >1.26E−05 >1.26E−05 SF-539 0.132 0.462 0.491 0.516 0.5060.435 0.110 109 117 113 92 −17  3.06E−06  8.85E−06 >1.26E−05 SNB-190.176 0.905 0.857 0.880 0.887 0.883 0.437 93 97 98 97 36 7.38E−06 >1.26E−05 >1.26E−05 SNB-75 0.501 1.051 0.925 1.049 1.134 0.7030.438 77 100 115 37 −13  8.54E−07  6.98E−06 >1.26E−05 U251 0.192 0.7990.789 0.792 0.803 0.680 0.061 98 99 101 80 −68  2.02E−06  4.37E−06 9.47E−06 Melanoma LOX IMVI 0.173 1.304 1.175 1.170 0.974 0.997 0.319 8988 71 73 13  3.03E−06 >1.26E−05 >1.26E−05 MALME-3M 0.476 0.859 1.0060.949 0.868 0.819 0.275 138 123 102 90 −42  2.51E−06  6.02E−06 >1.26E−05M14 0.123 0.613 0.602 0.635 0.592 0.549 0.136 98 105 96 87 3 3.45E−06 >1.26E−05 >1.26E−05 SK-MEL-2 0.388 0.704 0.746 0.714 0.7350.676 0.323 113 103 110 91 −17  3.03E−06  8.81E−06 >1.26E−05 SK-MEL-280.513 1.090 1.088 1.107 1.093 1.057 0.546 100 103 101 94 6 3.98E−06 >1.26E−05 >1.26E−05 SK-MEL-5 0.454 2.107 2.049 2.066 2.0251.748 0.460 96 97 95 78 0  2.90E−06 >1.26E−05 >1.26E−05 UACC-257 0.5961.142 1.149 1.128 1.165 1.078 0.814 101 97 104 88 40 7.78E−06 >1.26E−05 >1.26E−05 UACC-62 0.306 1.082 1.111 1.105 1.1081.051 0.355 104 103 103 96 6  4.10E−06 >1.26E−05 >1.26E−05 OvarianCancer IGROV1 0.291 1.679 1.828 1.932 1.743 1.710 0.536 111 118 105 10218  5.22E−06 >1.26E−05 >1.26E−05 OVCAR-3 0.240 1.066 1.073 1.055 1.0450.980 0.343 101 99 97 90 12  4.10E−06 >1.26E−05 >1.26E−05 OVCAR-5 0.4571.206 1.243 1.230 1.157 1.181 0.715 105 103 93 97 34 7.07E−06 >1.26E−05 >1.26E−05 OVCAR-8 0.173 1.340 1.250 1.245 1.1591.107 0.495 92 92 84 80 28  4.71E−06 >1.26E−05 >1.26E−05 SK-OV-3 0.3510.865 0.836 0.852 0.853 0.867 0.557 94 97 98 100 40 8.59E−06 >1.26E−05 >1.26E−05 Renal Cancer A498 0.400 0.678 0.691 0.6570.649 0.635 0.357 105 93 89 84 −11  2.90E−06  9.71E−06 >1.26E−05 CAKI-10.372 0.942 1.058 0.960 1.103 0.819 0.325 120 103 128 78 −13  2.59E−06 9.13E−06 >1.26E−05 RXE 393 0.743 1.243 0.860 1.114 1.290 1.077 0.776 2374 109 67 7 >1.26E−05 >1.26E−05 SN12C 0.157 0.546 0.533 0.529 0.5000.474 0.113 97 96 88 81 −28  2.44E−06  6.96E−06 >1.26E−05 TX-10 0.3551.027 1.014 1.002 1.042 0.999 0.727 98 96 102 9655 >1.26E−05 >1.26E−05 >1.26E−05 UO-31 0.124 0.765 0.799 0.816 0.8300.785 0.391 105 108 110 103 42  9.20E−06 >1.26E−05 >1.26E−05 ProstateCancer PC-3 0.187 0.419 0.426 0.399 0.390 0.342 0.179 103 91 88 67 −4 2.17E−06  1.10E−05 >1.26E−05 DU-145 0.384 1.151 1.081 1.389 1.194 1.1190.873 91 131 106 96 64 >1.26E−05 >1.26E−05 >1.26E−05 Breast Cancer MCF70.200 0.928 0.924 0.912 0.985 0.893 0.277 99 98 108 95 11 4.31E−06 >1.26E−05 >1.26E−05 NCI/ADR-RES 0.328 1.386 1.576 1.376 1.4251.298 0.801 118 99 104 92 45  9.69E−06 >1.26E−05 >1.26E−05 MDA-MB- 0.3040.644 0.635 0.651 0.674 0.667 0.314 97 102 109 107 3 4.43E−06 >1.26E−05 >1.26E−05 231/ATCC HS 578T 0.369 1.255 1.338 1.2961.219 1.300 0.742 109 105 96 105 42  9.44E−06 >1.26E−05 >1.26E−05MDA-MB-435 0.465 1.425 1.370 1.354 1.476 1.369 0.666 94 93 105 94 21 5.05E−06 >1.26E−05 >1.26E−05 MDA-N 0.348 1.471 1.499 1.481 1.374 1.3160.385 102 101 91 86 3  3.44E−06 >1.26E−05 >1.26E−05 BT-549 0.735 1.7621.910 1.865 1.847 1.599 0.998 114 110 108 84 26 4.82E−06 >1.26E−05 >1.26E−05 T-47D 0.464 1.007 0.941 1.054 1.184 0.9050.419 88 109 133 81 −10  2.78E−06  9.86E−06 >1.26E−05

In the first set of tests, according to Table 1 and the dose responsecurves as shown in FIG. 1A to 1I, arsenic trioxide was effective inreducing cell growth against almost all the cell lines tested. Inparticular, leukemic cell lines, melanoma cell lines, and ovarian cancercell lines responded consistently by showing a reduction of more than50% growth. According to the mean graph as shown in FIG. 2, most of theleukemic and melanoma cell lines, central nervous system cancer celllines SNB-75 and U251, prostate cancer cell line PC-3, renal cancer celllines A498, CAKI-1, SN12C, and lung cancer cell line NCI-H522 wereespecially sensitive (relative to all the cell lines tested) totreatment with arsenic trioxide. TABLE 2 Log 10 Concentration Time MeanOptical Densities Percent Growth Panel/Cell Line Zero Ctrl −8.9 −7.9−6.9 −5.9 −4.9 −8.9 −7.9 −6.9 −5.9 −4.9 GI50 TGI LC50 Leukemia CCRF-CEM0.047 3.511 3.601 3.592 3.051 0.320 0.048 103 102 87 8 0 3.68E−07 >1.26E−05 >1.261−05 K-562 0.041 3.011 3.042 2.992 3.360 2.9310.096 101 99 112 97 2  3.941−06 >1.26E−05 >1.26E−05 MOLT-4 0.018 0.5030.554 0.228 0.432 0.245 0.113 111 43 85 47 20 >1.26E−05 >1.26E−05RPMI-8226 0.066 1.432 1.607 1.263 1.423 0.368 0.065 113 88 99 22 −2 5.481−07  1.091−05 >1.26E−05 Non-small Cell Lung Cancer EKVX 0.0300.682 0.573 0.601 0.628 0.414 0.014 83 88 92 59 −53  1.51E−06  4.22E−06 1.18E−05 HOP-62 0.061 1.026 1.076 0.997 1.019 0.451 0.014 105 97 99 40−77  8.66E−07  2.781−06  7.41E−06 HOP-92 0.115 1.292 1.455 1.834 1.3191.229 0.079 114 146 102 95 −31  2.85E−06  7.111−06 >1.26E−05 NCI-H2260.094 0.498 0.504 0.458 0.489 0.348 0.164 101 90 98 63 17 2.40E−08 >1.261−05 >1.26E−05 NCI-H23 0.075 1.624 1.818 1.814 1.8721.303 0.086 113 112 116 79 1  2.97E−06 >1.261−05 >1.26E−05 NCI-H322M0.035 0.702 0.680 0.620 0.674 0.552 0.054 97 88 96 77 3 2.94E−06 >1.261−05 >1.26E−05 NCI-H460 0.034 2.839 2.797 2.806 2.7922.535 0.062 99 99 98 89 1  3.50E−06 >1.261−05 >1.26E−05 NCI-H522 0.1751.224 1.269 0.535 1.191 0.128 0.028 104 34 97 −27 −84  7.641−07 3.19E−06 Colon Cancer COLO 205 0.022 2.351 2.308 2.026 2.283 1.4040.024 98 86 97 59 0  1.81E−06 >1.26E−05 >1.26E−05 HCT-116 0.046 3.4203.395 3.289 3.381 3.284 1.931 99 96 99 9656 >1.26E−05 >1.26E−05 >1.26E−05 HCT-15 0.033 3.838 3.965 3.921 4.2273.432 0.404 103 102 110 89 10  3.93E−06 >1.26E−05 >1.26E−05 HT29 0.0173.060 3.189 2.988 2.706 1.476 0.027 104 98 88 48 0 1.12E−06 >1.26E−05 >1.26E−05 KM12 0.012 0.808 0.487 0.533 0.507 0.0360.017 80 87 83 4 1  3.29E−07 >1.26E−05 >1.26E−05 SW-620 0.023 2.6522.613 2.308 2.337 1.475 0.007 99 87 88 55 −72  1.39E−06  3.43E−06 8.50E−06 CNS Cancer SF-268 0.054 0.798 0.990 0.381 0.356 0.387 −0.004126 44 41 45 −100  1.06E−08  2.57E−06  5.69E−06 SF-295 0.031 1.324 1.2841.279 1.244 0.659 0.010 97 97 94 49 −69  1.17E−06  3.25E−06  8.64E−06SF-539 0.041 1.598 1.727 1.403 1.545 0.691 0.017 108 87 97 42 −59 8.91E−07  3.29E−06  1.04E−05 SNB-75 0.134 1.065 1.143 1.033 1.024 0.8150.028 108 97 96 73 −79  1.79E−06  3.80E−06  8.08E−06 U251 0.105 2.3242.129 2.018 1.908 0.088 −0.012 91 86 81 −17 −100  2.63E−07  8.51E−07 3.16E−06 Melanoma LOX IMVI 0.013 2.153 2.000 1.666 0.053 0.002 −0.00193 77 2 −88 −100  2.90E−08  1.32E−07  4.73E−07 MALMI-3M 0.115 0.6500.702 0.595 0.823 0.383 0.005 110 90 132 50 −96  1.26E−06  2.78E−06 6.12E−06 SK-MEL-2 0.213 0.500 0.491 0.462 0.426 0.128 97 87 74 −40 −100 2.06E−07  5.63E−07  1.85E−06 SK-MEL-28 0.093 2.030 2.012 1.906 1.7970.488 99 94 88 20 −100  4.60E−07  1.86E−06  4.84E−06 SK-MEL-5 0.1811.907 1.935 1.864 1.929 1.846 0.013 102 97 101 96 −93  2.22E−06 4.07E−06  7.46E−06 UACC-257 0.053 0.883 0.872 0.857 0.832 0.504 0.03999 97 94 54 −27  1.42E−06  5.83E−06 >1.26E−05 UACC-62 0.030 1.537 1.5461.460 1.409 0.539 0.001 101 95 92 34 −98  6.60E−07  2.27E−06  5.43E−06Ovarian Cancer IGROV1 0.335 3.115 3.541 3.056 3.204 2.877 0.216 115 98103 91 −36  2.67E−06  6.62E−06 >1.26E−05 OVCAR-3 0.081 1.153 1.047 1.1281.124 0.482 0.001 90 98 97 37 −99  7.76E−07  2.36E−06  5.49E−06 OVCAR-50.038 1.404 1.433 1.460 1.478 1.287 0.028 102 104 105 91 −26  2.83E−06 7.53E−06 >1.26E−05 OVCAR-8 0.016 2.090 2.272 1.848 2.242 1.437 −0.001109 88 107 68 −100  1.62E−06  3.21E−06  6.36E−06 SK-OV-3 0.041 1.4741.445 1.410 1.461 1.247 0.020 98 96 99 84 −51  2.25E−06  5.27E−06 1.23E−05 Renal Cancer 786-0 0.021 1.578 1.582 1.821 1.786 0.832 −0.003100 116 113 52 −100  1.30E−06  2.77E−06  5.91E−06 A498 0.051 0.741 0.8260.764 0.837 0.558 0.025 112 103 114 73 −52  1.94E−06  4.85E−06  1.22E−05CAKI-1 0.032 1.352 1.254 1.725 1.573 0.491 0.004 93 128 117 35 −88 8.22E−07  2.43E−06  6.22E−06 RXF 393 0.198 1.515 1.327 1.301 1.7270.909 0.017 86 84 116 54 −91  1.34E−06  2.96E−06  6.54E−06 S12C 0.0240.956 0.882 1.141 1.113 0.829 0.013 92 120 117 86 −46  2.37E−06 5.67E−06 >1.26E−05 TK-10 0.072 1.333 1.356 1.268 1.334 1.103 0.239 10295 100 82 13  3.66E−06 >1.26E−05 >1.26E−05 Prostate Cancer DU-145 0.0952.158 2.147 1.954 1.935 2.153 0.055 99 90 89 100 −43  2.82E−06 6.32E−06 >1.26E−05 Breast Cancer MCF7 0.028 2.650 2.647 2.611 2.6031.683 0.008 100 99 98 63 −73  1.57E−06  3.66E−06  8.51E−06 NCI/ADR-RIS0.050 2.304 2.075 2.253 1.951 1.319 0.072 90 98 84 56 1 1.64E−06 >1.26E−05 >1.26E−05 MDA-MB-231/A 0.158 0.761 0.759 0.764 0.7710.563 −0.001 100 101 102 67 −100  1.59E−06  3.18E−06  6.33E−06 TCC HS578T 0.109 0.728 0.777 0.587 0.890 0.706 0.010 108 77 126 96 −91 2.23E−06  4.12E−06  7.62E−06 MDA-MB-435 0.054 1.500 1.276 1.470 0.8650.631 0.021 85 98 56 40 −62  3.00E−07  3.10E−06  9.60E−06 MDA-N 0.0282.652 2.468 2.616 2.606 1.738 0.026 93 99 98 65 −9  2.02E−06 9.55E−06 >1.26E−05 BT-549 0.112 1.798 1.876 1.940 1.963 1.697 0.018 105108 110 94 −84  2.22E−06  4.24E−06  8.08E−06 T-47D 0.098 0.824 0.8830.336 0.539 0.398 0.027 108 33 61 41 −73  2.90E−06  7.93E−06

In the second set of tests, according to Table 2, the dose responsecurves, and the mean graph as shown in FIG. 3A to 3I, and FIG. 4,arsenic trioxide was effective in reducing cell growth against all thecell lines tested. The results were consistent with those obtained fromthe first set of tests. In particular, several melanoma cell linesappeared to be especially sensitive at the various principal responseparameters.

In conclusion, these results demonstrate that arsenic trioxide iseffective in inhibiting the growth of leukemic cells and cancer cells invitro, and that arsenic trioxide can be used in human subjects to treata broad range of leukemia, and cancers, including but not limited tonon-small cell lung cancer, colon cancer, central nervous system cancer,melanoma, ovarian cancer, renal cancer, prostate cancer, and breastcancer.

The present invention is not to be limited in scope by the specificembodiments described herein. Indeed, various modifications of theinvention in addition to those described herein will become-apparent tothose skilled in the art from the foregoing description. Suchmodifications are intended to fall within the scope of the appendedclaims.

1. A method of treating multiple myeloma in a human, which comprisesadministering a therapeutically effective amount of one or more arseniccompounds to said human.
 2. The method of claim 1, wherein said arseniccompound is arsenic trioxide.
 3. The method of claim 2, wherein saidarsenic trioxide is formulated as an ionic aqueous solution.
 4. Themethod of claim 1, wherein the total daily amount administered is fromabout 10 μg to about 200 mg.
 5. The method of claim 1, wherein the totaldaily amount administered is from about 0.5 mg to about 150 mg.
 6. Themethod of claim 1, wherein the total daily amount administered is fromabout 0.5 mg to about 70 mg.
 7. The method of claim 1, wherein thearsenic compound is administered parenterally.
 8. The method of claim 1,wherein the arsenic compound is administered intravenously.
 9. Themethod of claim 1, wherein the arsenic compound is administered incombination with an effective amount of at least one other therapeutic.10. The method of claim 9, wherein the other therapeutic agent is achemotherapeutic or radiotherapeutic.
 11. The method of claim 9, whereinthe other therapeutic agent is selected from the group consisting ofetoposide, cisplatin, carboplatin, estramustine phosphate, vinblastine,methotrexate, hydroxyurea, cyclophosphamide, doxorubicin,5-fluorouracil, taxol, diethylstilbestrol, VM-26(vumon), BCNU, all-transretinoic acid, procarbazine, cytokines, therapeutic vaccines, andimmunomodulators.
 12. The method of claim 3, wherein the dose is variedaccording to the body weight of said human.